A 3D-microtissue-based phenotypic screening of radiation resistant tumor cells with synchronized chemotherapeutic treatment.

BACKGROUND: Radiation resistance presents a challenge to the effective treatment of cancer. If therapeutic compounds were capable of resensitizing resistant tumours then a concurrent chemo-radiation treatment could be used to overcome radiation resistance. METHODS: We have developed a phenotypic assay to investigate the response of radiation resistant breast cancer cells grown in 3D-microtissue spheroids to combinations of radiation and established chemotherapeutic drugs. The effects were quantified by real time high content imaging of GFP detection area over 14 days. Ten established chemotherapeutic drugs were tested for their ability to enhance the effects of radiation. RESULTS: Of ten analysed chemotherapeutics, vinblastine was the most effective compound, with docetaxel and doxorubicine being less effective in combination with radiation. To investigate the response in a model closer to the in vivo situation we investigated the response of heterotypic 3D microtissues containing both fibroblasts and breast cancer cells. Drug treatment of these heterotypic 3D cultures confirmed treatment with radiation plus vinblastine to be additive in causing breast cancer growth inhibition. We have validated the screen by comparing radiation sensitizing effects of known chemotherapeutic agents. In both monotypic and heterotypic models the concurrent treatment of vinblastine and radiation proved more effective inhibitors of mammary cancer cell growth. The effective concentration range of both vinblastine and radiation are within the range used in treatment, suggesting the 3D model will offer a highly relevant screen for novel compounds. CONCLUSIONS: For the first time comfortable 3D cell-based phenotypic assay is available, that allows high throughput screening of compounds with radiation therapy modulating capacity, opening the field to drug discovery.

Streptomyces sp. JS520 produces exceptionally high quantities of undecylprodigiosin with antibacterial, antioxidative, and UV-protective properties.

A Gram-positive, red-pigment-producing bacterial strain, designated JS520 was isolated from the pristine sediment from the cave on mountain Miroc in Serbia. Strain was confirmed to belong to Streptomyces genus based on phenotypic and genetic analysis. Streptomyces sp. JS520 has the ability to produce exceptionally high amounts of deep red pigment into both solid and liquid media. Liquid chromatography and mass spectroscopy of the purified pigments revealed the major component to be undecylprodigiosin (93 %) with minor component being oxidatively cyclized derivative. The pigment production was affected by medium composition, temperature, pH, and the aeration rate. By medium optimization, yields of undecylprodigiosin of 138 mg l(-1) were achieved, what is the highest level of undecylprodigiosin production reported for the members of Gram-positive Streptomyces genus. Purified pigment had antimicrobial properties against bacterial Bacillus and Micrococcus species (50 µg ml(-1)) and against Candida albicans species (100-200 µg ml(-1) range). The ability to affect auto-oxidation of the linoleic acid was demonstrated for the purified undecylprodigiosin, suggesting antioxidative properties of this pigment. Multiple ecophysiological roles of the pigment were revealed by comparing cultures grown under pigment-producing and pigment-nonproducing conditions. Cells grown under undecylprodigiosin-producing conditions could tolerate presence of hydrogen peroxide exhibiting three times smaller zones of inhibition at 100 mM H(2)O(2). Undecylprodigiosin-producing cells were also less susceptible to tetracycline, kanamycin, chloramphenicol, and 8-hydroxyquinoline. While the growth of the cells not producing pigment was completely inhibited by 15 min of exposure to ultraviolet light (254 nm), cells producing undecylprodigiosin and cells supplied with purified pigment in vitro showed survival rates at 22 and 8 %, respectively.

Differential response of normal and transformed mammary epithelial cells to combined treatment of anti-miR-21 and radiation.

PURPOSE: MicroRNA miR-21 has emerged as a therapeutic target in the treatment of breast cancer. This study was designed to compare the responses of breast cancer cells and non-transformed breast epithelial cells to a combined regimen of miR-21 inhibition and radiation. MATERIALS AND METHODS: The MDA-MB-361 (breast cancer) and MCF-10A (non-transformed mammary epithelial) cell lines were used for the comparison in this in vitro study. The stable knockdown of miR-21 was performed by using lentiviral approach. The response of the cells was monitored 4, 24 and 48 h after the irradiation with 0.25 and 2.5 Gy, using sham-irradiated cells as controls. The response of the cells was established by performing various functional assays - cell viability and cell attachment, clonogenic survival, cell cycle analysis and 3D microtissue formation. RESULTS: The knockdown of miR-21 induced significant increase in apoptosis and growth delay in MDA-MB-361 cancer cells compared to non-transformed MCF-10A cells. After combined radiation and anti-miR-21 treatment, MDA-MB-361 cells show reduced cell growth and viability what is presented in their inability to form colonies. MCF-10A cells were not as sensitive to the combined treatment and that has also been confirmed with colony forming assay. CONCLUSIONS: Cellular response to a combined treatment of anti-miR-21 and radiation is different between cancer and non-cancer cells which highly support the idea of linking miR-21 inhibitor and radiation treatment in the future therapeutic approaches for breast cancer.

Radiation induced transcriptional and post-transcriptional regulation of the hsa-miR-23a~27a~24-2 cluster suppresses apoptosis by stabilizing XIAP.

The non-coding transcriptome, in particular microRNAs (miRNA), influences cellular survival after irradiation. However, the underlying mechanisms of radiation-induced miRNA expression changes and consequently target expression changes are poorly understood. In this study we show that a single dose of 5Gy ɣ-radiation decreases expression of the miR-23a~27a~24-2 cluster in the human endothelial cell-line EA.hy926 and the mammary epithelial cell-line MCF10A. In the endothelial cells this was facilitated through transcriptional regulation by promoter methylation and also at the post-transcriptional level by reduced miRNA processing through phosphorylation of Argonaute (AGO). Furthermore, we demonstrate that all three mature cluster miRNAs reduce apoptosis by increasing expression of the common target protein XIAP. These findings link a temporal succession of transcriptional and post-transcriptional regulatory mechanisms of the miR~23a~24-2~27a cluster, enabling a dynamic stress response and assuring cellular survival after radiation exposure.

Radiation alters the cargo of exosomes released from squamous head and neck cancer cells to promote migration of recipient cells.

Radiation is a highly efficient therapy in squamous head and neck carcinoma (HNSCC) treatment. However, local recurrence and metastasis are common complications. Recent evidence shows that cancer-cell-derived exosomes modify tumour cell movement and metastasis. In this study, we link radiation-induced changes of exosomes to their ability to promote migration of recipient HNSCC cells. We demonstrate that exosomes isolated from irradiated donor cells boost the motility of the HNSCC cells BHY and FaDu. Molecular data identified enhanced AKT-signalling, manifested through increased phospho-mTOR, phospho-rpS6 and MMP2/9 protease activity, as underlying mechanism. AKT-inhibition blocked the pro-migratory action, suggesting AKT-signalling as key player in exosome-mediated migration. Proteomic analysis of exosomes isolated from irradiated and non-irradiated BHY donor cells identified 39 up- and 36 downregulated proteins. In line with the observed pro-migratory effect of exosomes isolated from irradiated cells protein function analysis assigned the deregulated exosomal proteins to cell motility and AKT-signalling. Together, our findings demonstrate that exosomes derived from irradiated HNSCC cells confer a migratory phenotype to recipient cancer cells. This is possibly due to radiation-regulated exosomal proteins that increase AKT-signalling. We conclude that exosomes may act as driver of HNSCC progression during radiotherapy and are therefore attractive targets to improve radiation therapy strategies.

Secreted uPAR isoform 2 (uPAR7b) is a novel direct target of miR-221.

miR-221/-222 and components of the urokinase-type plasminogen activator system (uPAS) are associated with metastasis and poor prognosis in breast cancer, including the triple-negative subtype (TNBC). Modification of components of uPAS and involved miRNAs may contribute to targeted therapy for breast cancer patients. miR-221-/-222-overexpressing or miR-221-depleted cells were employed for qRT-PCR and Western blots to show associations of uPAR with miR-221/-222. To substantiate direct targeting of miR-221/-222 within 3' UTR of the uPAR isoform 2, in silico analysesand in vitro assays were conducted. Significant associations between miR-221 and uPAR isoform 2 expressions were observed at the mRNA and protein levels in breast cancer cells representing TNBC. For the first time, the uPAR isoform 2 was demonstrated as direct target for miR-221/-222. Inhibition of miR-221 reduced uPAR protein expression and expression of the tumor cell invasion markers vimentin and RHOC. These results demonstrate a direct and positive regulation of the secreted uPAR isoform 2 by miR-221, increasing its protein expression, a prerequisite for malignancy, while the other uPAR isoforms (1, 3 and 4) are indirectly regulated through miR-10b and miR-221/-222. By targeting uPAR isoforms and/or miRNA-221/-222, the diagnosis and therapy of breast cancer, in particular in TNBC, could be significantly improved.

PARTICLE, a Triplex-Forming Long ncRNA, Regulates Locus-Specific Methylation in Response to Low-Dose Irradiation.

Exposure to low-dose irradiation causes transiently elevated expression of the long ncRNA PARTICLE (gene PARTICLE, promoter of MAT2A-antisense radiation-induced circulating lncRNA). PARTICLE affords both a cytosolic scaffold for the tumor suppressor methionine adenosyltransferase (MAT2A) and a nuclear genetic platform for transcriptional repression. In situ hybridization discloses that PARTICLE and MAT2A associate together following irradiation. Bromouridine tracing and presence in exosomes indicate intercellular transport, and this is supported by ex vivo data from radiotherapy-treated patients. Surface plasmon resonance indicates that PARTICLE forms a DNA-lncRNA triplex upstream of a MAT2A promoter CpG island. We show that PARTICLE represses MAT2A via methylation and demonstrate that the radiation-induced PARTICLE interacts with the transcription-repressive complex proteins G9a and SUZ12 (subunit of PRC2). The interplay of PARTICLE with MAT2A implicates this lncRNA in intercellular communication and as a recruitment platform for gene-silencing machineries through triplex formation in response to irradiation.

Metabolic versatility of Gram-positive microbial isolates from contaminated river sediments.

Gram-positive bacteria from river sediments affected by the proximity of a petrochemical industrial site were isolated and characterized with respect to their ability to degrade a wide range of aromatic compounds. In this study we identified metabolically diverse Gram-positive bacteria capable of growth on wide range aromatic compounds in the presence of heavy metals and with the ability to accumulate biopolymers. Thirty-four isolates that were able to use 9 or more common aromatic pollutants, such as benzene, biphenyl, naphthalene etc. as a sole source of carbon and energy included members of Bacillus, Arthrobacter, Rhodococcus, Gordonia, Streptomyces, and Staphylococcus genus. Rhodococcus sp. TN105, Gordonia sp. TN103 and Arthrobacter sp. TN221 were identified as novel strains. Nine isolates were able to grow in the presence of one or more metals (mercury, cadmium, nickel) at high concentration (100mM). Seven isolates could degrade 15 different aromatic compounds and could grow in the presence of one or more heavy metals. Two of these isolates were resistant to multiple antibiotics including erythromycin and nalidixic acid. One third of isolates could accumulate at least one biopolymer. Twelve isolates (mainly Bacillus sp. and Arthrobacter sp.) accumulated polyphosphate, 3 Bacillus sp. accumulated polyhydroxybutyrate, while 4 isolates could accumulate exopolysaccharides.